UNIVERSITÀ DEGLI STUDI DI PADOVA
Department of Pharmaceutical and Pharmacological Sciences PhD Course in Pharmacological Sciences
Curriculum Pharmacology, Toxicology and Therapeutics
XXXI Cycle
Functional and Molecular Studies of the Crosstalk between Intestinal Microbioma and Enteric Nervous
System and Potential Effects on the Gut-Brain Axis
Coordinator: Ch.mo Prof. Piero Maestrelli
Supervisor: Ch.ma Prof.ssa Maria Cecilia Giron
PhD student: Ilaria Marsilio
2015-2018
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Index
RIASSUNTO ... 5
ABSTRACT ... 9
1. INTRODUCTION... 13
1.1 Enteric Nervous System ... 13
1.2 Toll-like Receptors ... 19
1.2.1 Toll-like Receptors in the Nervous System ... 25
1.3 Microbiota-Gut-Brain axis ... 29
1.4 Enteric Neurotransmission ... 30
1.4.1 Cholinergic Neurotransmission ... 32
1.4.2 Tachykinergic Neurotransmission ... 33
1.4.3 Serotonergic Neurotransmission... 34
1.4.4 Nitrergic Neurotransmission... 35
1.4.5 Purinergic Neurotransmission ... 36
1.4.6 Others Neurotransmission ... 37
1.5 Oxidized Phospholipids ... 38
1.6 Intestinal Microbiota ... 41
1.7 Serotonin in the Gut and Tryptophan Metabolism ... 46
1.8 Gut Barrier and Visceral Hypersensitivity ... 49
2. AIM ... 51
3. MATERIALS and METHODS ... 53
3.1 Mice……….53
3.2 Mice Treatments ... 53
3.3 Confocal Immunohistochemistry ... 54
3.3.1 Immunohistochemistry on Frozen Sections ... 54
3.3.2 Immunohistochemistry on Ileal Whole Mount Preparations ... 55
3.3.3 Acquisition and Analysis of Images ... 57
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3.3.4 Acetylcholinesterase and NADPH-diaphorase Biochemical Staining in Ileal
Whole Mount Preparations ... 58
3.4 In Vitro Contractility Studies ... 58
3.5 Gastrointestinal Transit Analysis ... 60
3.6 Intestinal Paracellular Permeability ... 60
3.7 Pellet Frequency and Fecal Water Content ... 61
3.8 RNA Isolation and Quantitative RT-PCR ... 61
3.9 HPLC Analysis of Tryptophan Metabolites ... 63
3.10 Statistical Analysis ... 63
3.11 Materials and Reagents ... 64
4. RESULTS ... 65
4.1 Toll-Like Receptor 4 in Murine Small Intestine ... 65
4.1.1 TLR4 Influences Ileal Morphology and ENS Architecture ... 65
4.1.2 Absence of TLR4 Impairs Gastrointestinal Motility ... 67
4.1.3 TLR4 Deficiency Affects Excitatory Neurotransmission ... 67
4.1.4 TLR4 Modulates Inhibitory Neurotransmission ... 70
4.1.5 TLR4 Absence Affects Purinergic Inhibitory Neurotransmission ... 73
4.2 TLR4 in Mouse Central Nervous System ... 74
4.2.1 TLR4 is Required for Sustaining Neuron and Glia Network in Murine Hippocampus ... 75
4.3 TLR2 and TLR4 Signaling Modulates Small Intestine Function ... 77
4.3.1 OxPAPC-mediated TLR2 and TLR4 Inhibition Alters the Architecture of the Myenteric Plexus of Juvenile Mice ... 78
4.3.2 OxPAPC Treatment Increases Excitatory Neuromuscular Contractility ... 79
4.3.3 OxPAPC Treatment Affects Inhibitory Neurotransmission ... 80
4.3.4 OxPAPC Treatment Influences Ileal SERT and 5-HT Receptors Expression.. ... 81
4.3.5 OxPAPC Treatment Modifies Serotonergic Neurotransmission ... 83
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4.3.6 OxPAPC-mediated Inhibition of TLR2 and TLR4 Impairs Tryptophan
Metabolism... 84
4.4 Microbiota-Gut Axis Regulates Serotonergic Neurotransmission... 85
4.4.1 Antibiotic-induced Microbiota Dysbiosis Affects Visceral Sensitivity .... 85
4.4.2 Antibiotic-induced Microbiota Dysbiosis Affects Tachykinergic Neurotransmission ... 86
4.4.3 Serotonin Neurotransmission Involves Ileum Relaxation-Response following Antibiotic-induced Microbiota Dysbiosis ... 88
4.4.4 Antibiotics Treatment Influences Ileal SERT and 5-HT Receptors Expression ... 89
4.4.5 Antibiotics-induced Microbiota Dysbiosis Affects Tryptophan Metabolism... 91
4.4.6 Antibiotic-induced Microbiota Dysbiosis Causes Morphological Abnormalities in the Architecture of the Myenteric Plexus ... 93
5. DISCUSSION ... 95
6. CONCLUSIONS ... 109
7. REFERENCES ... 111
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RIASSUNTO
L'interazione fra i costituenti della parete intestinale e la microflora commensale costituisce il principale artefice del mantenimento della barriera mucosale, della promozione dello sviluppo del tratto gastrointestinale (GI) e della modulazione delle funzioni GI, quali motilità, secrezione, immunità mucosale e sensibilità viscerale.
Un'alterata microflora è stata associata a disordini GI (malattia infiammatoria cronica intestinale, MICI e sindrome dell'intestino irritabile, IBS) mentre cambiamenti del microbiota intestinale durante le fasi dell’infanzia e dell’adolescenza, causati da infezioni o antibiotici, predispongono all'insorgenza di queste malattie. Inoltre, disfunzioni del sistema nervoso enterico (SNE) quali anomalie strutturali e/o variazioni nel contenuto di neurotrasmettitori, sono state associate all'insorgenza sia di MICI che di IBS. In questo contesto, giocano un ruolo chiave i recettori Toll-like (TLRs), un sofisticato sistema di proteine che attivano la risposta immunitaria innata contro agenti patogeni e mediano segnali benefici al fine di assicurare l'integrità funzionale e strutturale sia in condizioni fisiologiche che patologiche. Polimorfismi nei geni che codificano i TLRs sono stati associati a fenotipi diversi di malattia in pazienti affetti da disordini GI. In questo studio sono state caratterizzate le alterazioni strutturali e funzionali del SNE murino indotte da:
i) cambiamenti nel segnale dell’immunità innata, mediato dal recettore TLR4, ii) una miscela di fosfolipidi ossidati (OxPAPC), implicati nel blocco del segnale generato dai recettori TLR2 e TLR4 al fine di eliminare parzialmente gli effetti mediati dalla flora intestinale batterica e iii) anomalie nella composizione del microbiota.
Data l’importanza di un corretto segnale TLRs-dipendente nel mantenimento della rete
nervosa e del codice neurochimico del SNE è stata valutata la funzione intestinale in vitro
mediante esperimenti di contrattilità utilizzando la tecnica dell'organo isolato su segmenti
di ileo provenienti da topi WT e TLR4
-/-di pari età (9 ± 1 settimane). Queste analisi hanno
evidenziato anomalie nell’attività contrattile neuromuscolare associate ad un’eccessiva
modulazione inibitoria controllata da ossido nitrico ed ATP, a sostegno della presenza di
un dialogo tra TLR4, SNE e microflora, fondamentale per la modulazione della funzione
neuromuscolare. Studi strutturali su preparati di ileo provenienti da topi TLR4
-/-hanno
dimostrato un’alterata architettura del SNE determinata da un’anomala distribuzione
della proteina gliale strutturale GFAP (glial fibrillary acidic protein) e della subunità β
della proteina S-100, marcatore gliale nucleare e citoplasmatico in grado di legare il
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calcio. Tali osservazioni indicano un coinvolgimento del recettore TLR4 nel mantenimento dell'integrità della rete gliale enterica mediato dalla produzione di ATP e nell’attivazione della trasmissione purinergica e pertanto evidenziano il ruolo primario di questo recettore nella conservazione dell'omeostasi strutturale e funzionale del SNE.
Inoltre, in tale modello, è stato approfondito il ruolo del recettore TLR4 nell’asse
‘intestino-cervello’ attraverso la valutazione strutturale del sistema nervoso centrale in particolare a livello dell’ippocampo, area deputata all’apprendimento. Negli animali TLR4
-/-è stato dimostrato che la mancanza del recettore TLR4 determina nell’ippocampo, come a livello del SNE, una compromessa neuroplasticità caratterizzata da alterazioni nella densità neuronale associata a variazioni della distribuzione della rete gliale, a confermare un ruolo fondamentale del segnale TLRs anche a livello centrale.
In parallelo, è stato ulteriormente indagato il ruolo primario del segnale mediato dai recettori TLRs nell’asse microbiota-TLRs-SNE, saggiando l’effetto di una somministrazione in acuto per 3 giorni consecutivi con OxPAPC, inibitore del segnale mediato da entrambi i recettori TLR2 e TLR4, in topi adolescenti (3 ± 1 settimane). Il trattamento con OxPAPC ha causato un’alterazione significativa della risposta neuromuscolare sia recettore-mediata che non, nei topi trattati rispetto al controllo, associata a modifiche della rete neuro-gliale del SNE, confermando l’importanza del segnale mediato da tali recettori nell'assicurare l’integrità funzionale e strutturale del SNE durante l'adolescenza. Recenti studi riportano un ruolo primario nel dialogo tra i recettori TLRs e il sistema serotoninergico, spesso coinvolto in disturbi intestinali, pertanto è stato valutato se la somministrazione di OxPAPC per via intraperitoneale influenzasse tale sistema. È stato evidenziato come l’inibizione in acuto del segnale TLR2 e TLR4 comporti iperesponsività alla serotonina, alterazioni nella distribuzione recettoriale serotoninergica associata a variazioni nel metabolismo del triptofano, amminoacido coinvolto nella produzione di serotonina, a sostegno della presenza di un dialogo tra immunità innata e sistema serotoninergico.
Al fine di approfondire il ruolo dell'asse microbiota-intestino nell’omeostasi del SNE è
stato messo a punto un modello animale di deplezione di microbiota intestinale attraverso
la somministrazione intragastrica di 4 antibiotici, ampicillina (100 mg/kg), metronidazolo
(100 mg/kg), neomicina (100 mg/kg) e vancomicina (50 mg/kg) due volte al giorno per
14 giorni a topi C57BL/6J adolescenti (3 ± 1 settimane; topi ABX). Da una prima
valutazione il trattamento antibiotico ha determinato un fenotipo simil germ-free, come
già dimostrato da altri autori, ed alterazioni della motilità intestinale e dell'integrità della
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rete neuronale e gliale enterica. A tal proposito, analisi immunoistochimiche su preparati
di ileo provenienti da topi ABX hanno evidenziato anomalie nella distribuzione ed
espressione della proteina marcatore pan-neuronale HuC/D, della proteina gliale
strutturale GFAP e della subunità β della proteina S-100. Data l’importanza di una
corretta composizione del microbiota commensale sia nel mantenimento della rete
nervosa e del codice neurochimico del SNE che nella produzione di neurotrasmettitori a
livello enterico, sono state studiate le vie di neurotrasmissione coinvolte nella sensibilità
viscerale in tale modello di disbiosi intestinale. È stato osservato un incremento dei livelli
di mRNA di GluN1 e TRPV1 nel plesso mienterico dei preparati di ileo provenienti dai
topi ABX, evidenziando gli effetti di un’alterata composizione del microbiota intestinale
sulla sensibilità viscerale. Infine, è stato valutato l’effetto di uno stato di disbiosi indotto
da antibiotici sul sistema serotoninergico, sistema le cui funzioni sono modulate da
serotonina, metabolita la cui produzione è influenzata dall’azione di specifiche spore
batteriche. Il trattamento antibiotico riporta anomalie nella risposta neuromuscolare alla
serotonina accompagnate da una compromessa rete recettoriale serotoninergica e del
metabolismo del triptofano, sottolineando l’importanza di una corretta composizione del
microbiota nel mantenimento delle funzioni mediate dal sistema serotoninergico.
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ABSTRACT
The interaction between cellular constituents of gastrointestinal (GI) tract and commensal microflora is essential for the maintenance of mucosal barrier, promotion of the development of the GI system and modulation of enteric functions such as motility, secretion, mucosal immunity and visceral sensitivity. Alterations in the composition of the gut microflora have been associated to several GI disorders (e.g. inflammatory bowel disease, IBD, and irritable bowel syndrome, IBS) while changes in intestinal microbiota during infancy and adolescence, caused by infection or antibiotic therapy, appear to predispose to the onset of these diseases. Furthermore, dysfunctions of the enteric nervous system (ENS) such structural abnormalities and/or changes in the content of neurotransmitters, have been associated with the onset of IBD and IBS. In this context, a sophisticated system of proteins, so-called Toll-like receptors (TLRs), plays a key role in mediating the inflammatory response against pathogens and triggers beneficial signals to ensure tissue integrity under physiological and pathological conditions. Polymorphisms in genes encoding TLRs, including TLR2 or TLR4, have been associated with different phenotypes of disease extent and severity in patients with GI disorders. In this study we characterized structural and functional alterations of murine ENS induced by: i) changes in innate immunity response, mediated by TLR4, ii) a mixture of oxidized phospholipids (OxPAPC) that blocks both TLR2 and TLR4 signaling to partially avoid the recognition of gut commensal microflora and iii) anomalies in the composition of the microbiota.
Highlighted the role of proper TLRs signaling in the maintenance of neuronal network and neurochemical coding of the ENS, intestinal contractility was evaluated in isolated ileal segments from WT e TLR4
-/-mice (9±1 weeks) using organ bath technique.
Functional studies reported significant alterations of intestinal contractility associated to
an increased inhibitory neurotransmission via the combined action of nitric oxide (NO)
and adenosine-5′-triphosphate (ATP), suggested a crosstalk between TLR4, ENS and
microflora in the fine-tuning of ileal contractility. Furthermore, the absence of TLR4
affects ENS architecture characterized by abnormalities in the distribution and expression
of the pan-neuronal marker HuC/D and induced a reactive gliosis state with alterations in
the glial structural protein GFAP (glial fibrillary acidic protein) and the cytoplasmatic
and nuclear glial calcium-binding protein S100β in the ileal myenteric plexus. Once
demonstrated that TLR4 signaling is involved in the control of purinergic pathways in
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enteric neural-glial communication and highlighted its role in tuning structural and functional integrity of ENS, we assessed the role of TLR4 receptors in the central nervous system (CNS), in particular in the hippocampus assessing few architectural proteins expressed in neurons or astrocytes or microglial cells. The absence of TLR4 receptor determines neuroplasticity in the hippocampus, as well as in the ENS, characterized by a reduction of neuronal density associated with altered glial networks, to underline a key role of TLRs also in the CNS.
In parallel, to investigate the importance of TLRs-dependent signaling in modulating ENS-microbiota axis, juvenile male C57BL/6J mice (3±1 weeks old) were treated intraperitoneally with OxPAPC, that blocks both TLR2 and TLR4 signaling, twice a day for 3 days. In vivo inhibition of both TLR2 and TLR4 determined a significant alteration of receptor and non-receptor-mediated neuromuscular responses and affected myenteric plexus integrity, providing evidence that TLR2 and TLR4 signaling is essential in ensuring the structural and functional integrity of the ENS during adolescence. Recent studies demonstrated the role of TLRs in modulating intestinal serotonergic system and given that this system is involved in many GI functions, we evaluated the effect of OxPAPC treatments in this context. OxPAPC-mediated TLR2 and TLR4 inhibition affects serotonin-mediated response, in term of hyperresponsivity, and alters both serotonergic receptor distributions and tryptophan (TRP) metabolism during adolescence suggesting a cross-talk between innate immunity and serotonergic system.
To investigate the role of the microbiota-gut axis in the homeostasis of ENS we depleted gut microbiota by intragastric administration of a cocktail of broad spectrum antibiotics (50 mg/kg vancomycin, 100 mg/kg neomycin, 100 mg/kg metronidazol and 100 mg/kg ampicillin) twice a day for 14 days in adolescent mice (aged 3 ± 1 weeks, ABX). Mice after antibiotic treatment displayed a phenotype-like germ-free mice, as already reported by other Authors, and reveled an impairment in intestinal motility and in the neuro-glia integrity. Immunohistochemical analysis of ileal preparations from ABX mice showed abnormalities in the distribution and expression of the pan-neuronal marker HuC/D, the glial proteins GFAP and S100β. Given the importance of proper composition of commensal microbiota in the maintenance of neuronal network and neurochemical coding of the ENS and in the influencing neurotransmitter content, it has been investigated enteric neurotransmission involved in the control of central sensitization.
Increased mRNA levels of GluN1 and TRPV1 in the myenteric plexa of ABX mice was
found, suggesting that commensal microbiota is involved in modulating visceral
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sensitivity. Finally, the effect of antibiotic mediated microbiota dysbiosis in serotonergic
system was evaluated. The concept of a direct communication between commensals and
the enteric nervous system was suggested by different Authors; specifically, indigenous
spore-forming bacteria from mouse and human microbiota have been shown to promote
serotonin biosynthesis. The antibiotic treatment affects serotonin-mediated response
associated with impairments of serotonergic pathways and TRP metabolism, to evidence
an involvement of microbiota in serotonin-mediated functions and potentially in
microbiota-gut axis.
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1. INTRODUCTION
1.1 Enteric Nervous System
The enteric nervous system (ENS) has received special attention in the last years since it is the only limb of the peripheral nervous system (PNS) which has the ability to function independently from the central nervous system (CNS) and as such it has often been referred to as the “second brain” or the “little brain” (Goyal & Hirano, 1996).
The ENS is a complex tissue, extending from the esophagus to the anal sphincter within
the gastrointestinal (GI) system walls, and is composed of ganglia with neuronal fibers
innervating the effector tissues (Furness et al., 2014). The human ENS contains 200–600
million neurons, the same number of neurons that is found in the human spinal cord
(Furness & Costa, 1987a; Furness, 2006). The nerve-cell bodies are grouped into small
ganglia which are connected by bundles of nerve processes to form the two major
plexuses, so-called the myenteric (or Auerbach’s) plexus and the submucous (or
Meissner’s) plexus (Figure 1.1). A few small ganglia have been detected in the mucosa,
close to the muscularis mucosae (mucous plexus) (Hansen, 2003).
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Figure 1.1. Anatomy of ENS. (A) In the small and large intestines, neurons are confined in ganglia of the myenteric plexus (MP), localized between the longitudinal (LM) and circular muscle (CM) layers, and in ganglia distributed between the circular muscle and the muscularis mucosa (MM) within the submucosa (SMuc), depicted in the transverse section of the gut wall. The ganglia and fibers in the submucosa form inner and outer submucosal plexus (SMP). (B) The distribution of ganglia along the gastrointestinal tract.
(C) Neuromuscular layers along the small and large intestines (modified from Furness, 2012).
The myenteric plexus is positioned between the outer longitudinal and inner circular muscle layers, where forms a continuous network of ganglia that extends from the upper esophagus to the internal anal sphincter (Furness, 2012). It primarily provides motor innervation to the two muscle layers and secreto-motor innervation to the mucosa. There are numerous projections from the myenteric plexus to the submucosal ganglia and to enteric ganglia of the gallbladder and pancreas (Kirchgessner & Gershon, 1990).
Moreover, a substantial number of projections from the myenteric neurons are connected to the sympathetic ganglia (Goyal & Hirano, 1996; Hansen, 2003; Figure 1.1). The myenteric plexus shows a high density of neurons compared to the submucous plexus with an average ratio of the sensory, interneurons and motor neurons of 2:1:1, respectively (Costa et al., 2000; Hansen, 2003). In large mammals, the submucous plexus is located in the submucosa and composed by an inner network located at the serosal side of the muscularis mucosae (Meissner’s plexus) and an outer layer (Schabadasch’s plexus) adjacent to the luminal side of the circular muscle layer. Moreover, in the human intestine, a third intermediate plexus lies between Meissner’s and Schabadasch’s plexus. Non- ganglionated plexuses also supply all the layers of the gut (Costa et al., 2000; Furness, 2000; Hansen, 2003). Submucosal ganglia and connecting fiber bundles form plexuses in the small and large intestines, but these ganglia are extremely rare in the stomach and esophagus (Furness, 2012; Figure 1.1).
(C)
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The ENS is in continuous communication with autonomic nervous system (ANS) through sympathetic and parasympathetic afferent and efferent neurons. The ANS drives both afferent signals, arising from the lumen and transmitted through enteric, spinal and vagal pathways to CNS, and efferent signals from CNS to the intestinal wall. In the GI tract, sympathetic, parasympathetic, and spinal afferent nerve fibers are extrinsically innervating the ENS and ensure the bidirectional communication with the CNS through intimate connections with the spinal cord. The gut vast innervations and connections between intrinsic and extrinsic fibers guarantee the CNS monitoring of a number of gut parameters, from chemical sensing in the lumen, to sensing mechanical stress along the gut wall (Furness, 2000). Along the GI tract, the vagus nerve has three afferent endings within the gut wall: intraganglionic laminar endings within the myenteric plexus, intramuscular arrays within the smooth muscle layers and mucosal fibers within the mucosa. The stomach has the highest density of the vagal afferent ending and the density deceases towards the distal regions of the GI (Powley & Phillips, 2002). The sympathetic neurons (effector branch of the ANS) have axons that extend along the mesenteric nerves deep into the gut wall to the myenteric, submucosal and mucosal plexuses of the ENS (Lomax et al., 2010). The terminals of these axons are responsible of releasing numerous neurotransmitters, mainly norepinephrine (NE) and tyrosine hydroxylase (TH). In the other side, vagal efferent neurons of the motor pathways are parasympathetic preganglionic neurons (Hansen, 2003). A variety of central effects, primarily on the upper GI tract, are mediated through these neurons, including relaxation of the proximal stomach, enhancement of gastric peristalsis, and promotion of gastrin secretion.
Transmission from vagal input neurons to enteric neurons is mediated principally by acetylcholine (ACh) acting on nicotinic cholinergic receptors, but several other transmitters are involved in these processes (Hansen, 2003). This bidirectional connection, the so-called gut-brain axis, provides neural control of all functions of the GI tract (Goyal & Hirano, 1996). The ENS is endowed with a wide array of restorative, maintenance and adaptive functions. Motility patterns, gastric secretion, transport of fluid across the epithelium, blood flow, nutrient handling, interaction with the immune and endocrine systems of the gut are function under the control of the ENS (Furness, 2012;
Wood, 2012).
According to neurons morphology, neurochemical coding, cell physiology, projections to
targets and functional roles, approximately 20 distinct types of neurons have been
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described (Costa et al., 2000). The enteric neuronal circuits are composed by intrinsic primary afferents neurons, sensory neurons which detect mechanical distortion of the mucosa, mechanical forces in the external musculature (tension of the gut wall) or the presence of chemical luminal stimuli and initiate appropriate reflex control of functions including motility, secretion and blood flow (Clerc et al., 2002). Along the whole GI tract, the longitudinal and circular smooth muscle layers and the muscularis mucosae are innervated by uni-axonal excitatory and inhibitory motor neurons (Dogiel type I morphology), which receive prominent fast excitatory synaptic potentials (Wood, 2012).
The primary neurotransmitters for excitatory motor neurons are ACh and tachykinins.
Several neurotransmitters have been identified in inhibitory motor neurons, including nitric oxide (NO), vasoactive intestinal peptide (VIP) and adenosine triphosphate (ATP)- like transmitters, although NO is considered the primary transmitter (Furness et al., 2014;
Table 1.1).
Table 1.1. Proportions of all neurons attributed to different functional classes in myenteric ganglia of mouse small intestine (modified from Qu et al., 2008; Hao et al., 2013). Abbreviations: AH, after-hyperpolarizing;
S, synaptic.
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Another important class of enteric neurons is represented by secretomotor and secretomotor/vasodilator neurons regulating the electrolyte and water transport across the intestinal mucosa (Vanner & Macnaughton, 2004).
In the ENS, in parallel with neuron population, it is possible to identify another cell population that is represent by enteric glial cells (EGCs). In the last couple of years, the role of EGCs in ENS function has gained significant attention (Sharkey, 2015). EGCs constitute a major population of peripheral glia that is located within the ganglia of the myenteric and submucosal plexus of the ENS and in extraganglionic sites, such as the smooth muscle layers and the mucosa (Gershon & Rothman, 1991; Gulbransen &
Sharkey, 2012; Ruhl et al., 2004). The EGCs are usually small cells with highly irregular, stellate-shaped body, associated to neuronal cell bodies in enteric ganglia in an intimate physical connection, highly reminiscent of the relationship between astrocytes and neurons in the CNS (Gulbransen & Sharkey, 2012; Figure 1.2). EGCs also show connections with enteric nerve fiber bundles, which are similar to peripheral Schwann cells, but differ from these by the function (Lomax et al., 2005). Different types of EGCs have been identified (Boesmans et al., 2015; Hanani & Reichenbach, 1994) and are subdivided into four subtypes which correspond to unique locations within the plexus and extraganglionic spaces and to specific phenotypic properties (Boesmans et al., 2015). The EGCs ‘type I’ or ‘protoplasmic’ display star-shaped cells with short, irregularly branched processes resembling protoplasmic astrocytes of the CNS and closely embrace neuronal cell bodies and fibers within myenteric and submucosal ganglia (intraganglionic EGCs).
Enteric glia ‘type II’ represents the elongated glial cells within interganglionic fiber tracts, which are similar to fibrous astrocytes of the white matter in the CNS. The subepithelial glia consists of several long branches that reach the mucosal epithelial cells, and thus could be grouped as ‘mucosal’ or ‘type III’ EGCs. The fourth type of enteric gliocytes are distributed between smooth muscle cells, running with neuronal fibers in the musculature, thus these cells are ‘intramuscular’ or ‘type IV’ EGCs (Hanani &
Reichenbach, 1994; Figure 1.2). Traditionally, EGCs were thought to contribute
primarily to the structural integrity and nourishment of the ENS. However, during the last
decades several studies have confuted the concept of a merely supportive function of
EGCs and ascribed to them a wide variety of roles that are essential for proper GI function
(Boesmans et al., 2015). In addition to be a scaffold for neurons, EGCs are involved in
most gut functions such as mucosal integrity, neuroprotection, adult neurogenesis, neuro‐
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immune interactions, and synaptic transmission (De Giorgio et al., 2012; Gulbransen &
Sharkey, 2012; Neunlist et al., 2013; Ruhl et al., 2004).
Figure 1.2. Subpopulations of enteric glia. (A) Several subpopulations of enteric glia located within the gut wall with different proposed physiological functions and signaling mechanisms. (B) Mucosal enteric glia lies in the mucosa directly beneath the epithelial cells. (C) Intraganglionic glia surround neurons (blue) within the enteric nerve plexuses (submucosal and myenteric plexus). (D) Intramuscular glia is associated with enteric nerve fibers innervating the smooth muscle layers (circular muscle and longitudinal muscle).
Abbreviations: α2-AR, α2 adrenergic receptor; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; GAT2, sodium- and chloride-dependent GABA transporter 2; mGluR5, metabotropic glutamate receptor 5;
NTPdase2, ectonucleoside triphosphate diphosphohydrolase 2; PAR1/2, protease-activated receptor 1/2;
PEPT2, peptide transporter 2 (also known as solute carrier family 15 member 2); proEGF, proepidermal growth factor; P2X7, P2X7 receptor; P2Y1,2,4, P2Y1,2,4 receptor; TGF-β, transforming growth factor β.
(modified from Gulbransen & Sharkey, 2012).
Although at present EGCs are the least-studied peripheral glial cells in mammals, there
is an increasing interest in understanding the complex roles of these cells in GI
physiology. Clonal cultures of ENS progenitors have shown that EGCs originate from
common neuro-glial progenitors (Bondurand et al., 2003), but the presence of bi-potential
or committed neurogenic and gliogenic progenitors in vivo has not been documented so
far. Moreover, it remains unclear the role of individual progenitors in generating distinct
subtypes of enteric neurons and glial cells. In rodents, a significant fraction of enteric
neurons and EGCs develops during the early postnatal period, and it would be very
interesting to explore how changes associated with feeding or the establishment of
luminal microflora and the maturation of the mucosal immune system after birth affect
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ENS development (Kabouridis & Pachnis, 2015). Recently, emerging evidence suggests that gut microflora can have dramatic effects on the development and function of the nervous system, both at the local as well as at the systemic level (Obata & Pachnis, 2016).
The role of microbiota on ENS organization is highlighted by the reduced number of enteric neurons and the associated deficits in gut motility observed in germ-free (GF) mice (Anitha et al., 2013). Furthermore, the development and continuous homeostatic influx of EGCs into the intestinal mucosa is defective in GF mice or in antibiotic-treated mice (Kabouridis et al., 2015). These findings reveal the complex and intricate relationship between the microbiota and EGCs as regulators of neuroimmune control of host defense in the intestinal mucosa (Sharkey et al., 2018) and essential for the assembly of intestinal neural-glial circuits. Interestingly, reconstitution of GF mice with conventional microbiota normalized the density of EGCs network and gut physiology (Kashyap et al., 2013; Kabouridis et al., 2015) raising interesting questions relating to the cellular plasticity of the ENS and the mechanisms by which microbiota influence its homeostasis. Furthermore, the potential role of the microbiota and the mucosal immune system in the activation of glial progenitors and the homeostasis of EGCs is currently unclear, but it is interesting that glial cells are capable to direct influence immune responses (Turco et al., 2014). To this concern, an upon bacterial stimulation, EGCs upregulate expression of MHC class II, which suggests that they actively respond to the colonization of the gut lumen by microbiota and participate in antigen presentation to the adaptive immune system (Turco et al., 2014). Taken together, these observations highlight that dynamic host–microbe interactions are a key element for EGCs development, suggesting that an improved understanding of this mechanism will provide important insights into the pathophysiology of GI diseases.
1.2 Toll-like Receptors
All living organisms are constantly exposed to environmental microorganisms and cope
with their potential invasion into the body. The vertebrate immune response can be
divided into innate and acquired immunity. The innate immune system is the first line of
host defense against pathogens and is mediated by phagocytes including macrophages
and dendritic cells (DCs; Akira et al., 2006). In fact, to control the infection during the
first days, the organism, through innate immune system, modulates some important
functions including opsonization, activation of complement, coagulation cascades,
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phagocytosis, activation of proinflammatory signaling cascades and apoptosis (Janssens
& Beyaert, 2003). By contrast, acquired immune responses are slower processes, in the late phase of infection, which are mediated by T and B cells, both of which express highly diverse antigen receptors that are generated through DNA rearrangement and are thereby able to respond to a wide range of potential antigens and to generation of immunological memory (Akira et al., 2006). This highly sophisticated system of antigen detection is found only in vertebrates and has been the subject of considerable research. Far less attention has been directed towards innate immunity, as it has been regarded as a relatively nonspecific system, however is able to discriminate between self and non-self, such as a variety of pathogens and to present antigen to the cells involved in acquired immunity (Akira et al., 2006). Also, the innate immune system has an important function in activation and shaping of the adaptive immune response through the induction and release of co-stimulatory molecules and cytokines (Medzhitov, 2007; Figure 1.3). In contrast to the clonotypic receptors, expressed by B and T lymphocytes, the innate immune system uses nonclonal sets of recognition molecules, called pattern recognition receptors (PRRs; Janssens & Beyaert, 2003; Figure 1.3).
Figure 1.3. Pathways of host-defense mechanisms (modified from Medzhitov, 2007). Abbreviation: PRRs, pattern recognition receptors.
Toll like receptors (TLRs) are one of the most important family of the PRRs. The
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discovery of the TLRs started with the identification of the receptor ‘Toll’, a protein expressed in Drosophila melanogaster and involved in controlling embryonic development (Akira & Takeda, 2004; Okun et al., 2011). Subsequent genetic studies have led to the discovery of genes important in the dorsal-ventral patterning of the embryo (i.e., the dorsal group of genes, including Toll, tube, pelle, cactus, the NF-κB homolog dorsal, and seven genes upstream of Toll; Belvin & Anderson, 1996). Since NF-κB is involved in mammalian immunity, gradually became evident the contribution of TLRs in the signaling pathways in regulating Drosophila embryonic development and activating the immune system (Wasserman, 1993). In the 1995, Hultmark and colleagues first identified Toll-1 as an activator of the immune response in a Drosophila cell line. Around the same time, a human homolog of Toll was identified and mapped to chromosome 4p14 (Taguchi et al., 1996). Later on, an in vivo study in Drosophila demonstrated that the Toll signaling is involved also in the antifungal response (Lemaitre et al., 1996). In the 1997, the first mammalian TLRs was described by the group of Medzhitov. Subsequently, five human TLRs have been characterized (Rock et al., 1998) that are involved only in controlling immune responses with no role in the development whereas the Drosophila Toll pathway is implicated both in immunity and developmental processes (Valanne et al., 2011). TLRs are type I transmembrane proteins responsible in the recognition of foreign pathogens referred to as pathogen-associated molecular patterns (PAMPs). PAMPs are well suited to innate immune recognition for three main reasons: i) they are invariant among microorganisms of a given class; ii) they are products of pathways that are unique to microorganisms, allowing discrimination between self and non-self-molecules; iii) they have essential roles in microbial physiology, limiting the ability of the microorganisms to evade innate immune recognition through adaptive evolution of these molecules (Medzhitov, 2007). Bacterial PAMPs are often components of the cell wall, such as lipopolysaccharide (LPS), peptidoglycan (PG), lipoteichoic acids (LTA) and cell-wall lipoproteins. An important fungal PAMP is beta-glucan, which is a component of fungal cell walls, but also viral nucleic acids structures are recognized by TLRs. An important aspect of pattern recognition is that PRRs themselves do not distinguish between pathogenic microorganisms and symbiotic (non-pathogenic) microorganisms, because the receptor ligands are not unique to pathogens (Medzhitov, 2007). So far, 10 and 12 functional TLRs have been identified in humans and mice, respectively, with TLR1–
TLR9 being conserved in both species. Mouse TLR10 is not functional for a retrovirus
insertion, and TLR11, TLR12 and TLR13 have been lost from the human genome (Kawai
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& Akira, 2010; Table 1.2). Studies in mice deficient in each single TLRs type have demonstrated that every TLR has a distinct function in terms of PAMPs recognition and activation of immune responses (Akira et al., 2006).
TLR1, 2, 4 and 6 recognize lipid-based structures. TLR4 recognizes LPS from Gram- negative bacteria, which causes septic shock (Akira et al., 2006). TLR2 forms heterodimers with TLR1 and TLR6 and in concert with TLR1 or TLR6 discriminates between the molecular patterns of triacyl and diacyl lipopeptide, respectively, which derived from Gram-positive bacteria, mycoplasma and mycobacteria (Kumar et al., 2009). TLR5 and 11 recognize protein ligands. TLR5 is expressed abundantly in intestinal CD11c-positive lamina propria cells where it senses bacterial flagellin (Uematsu &
Akira, 2006). TLR3, 7, 8 and 9, being localized intracellularly, detect nucleic acids
derived from viruses and bacteria. TLR3 was shown to recognize double stranded RNA
(dsRNA) generally produced by many viruses during replication. TLR7 recognizes
synthetic imidazoquinoline-like molecules, guanosine analogs such as loxoribine, single
stranded RNA (ssRNA) derived from viruses and small interfering RNA (Akira et al.,
2006; Table 1.2). TLRs are expressed on a variety of cells, including immune cells, such
as macrophages, DCs, B cells, specific types of T cells, and also fibroblasts, epithelial
cells and neurons. Expression of TLRs is not static but rather is modulated rapidly in
response to pathogens, an array of cytokines and environmental stressors (Akira et al.,
2006). Furthermore, TLRs may be expressed extracellularly or intracellularly. While
certain TLRs (TLRs 1, 2, 4, 5, and 6) are expressed on the cell surface, others (TLRs 3,
7, 8, and 9) are found almost exclusively in intracellular compartments such as
endosomes, and their ligands, mainly nucleic acids, require internalization to the
endosome before receptor signaling is possible (Akira et al., 2006).
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Table 1.2. Descriptions of TLR location and characteristics (modified from Kumar et al., 2009; Duffy &
O'Reilly, 2016).
The engagement of TLRs by microbial components triggers the activation of signaling cascades, leading to the induction of genes involved in antimicrobial host defense. TLRs are characterized by an ectodomain composed of leucine rich repeats (LRR) that are responsible for recognition of PAMPs and a cytoplasmic domain homologous to the cytoplasmic region of the IL-1 receptor, known as the TIR domain, which is required for downstream signaling (Kawai & Akira, 2007).
After ligand binding, TLRs dimerize and undergo conformational changes required for
the recruitment of TIR-domain-containing adaptor molecules of the TLR (Akira et al.,
2006). The adaptor molecules include myeloid differentiation factor 88 (MyD88), TIR-
associated protein (TIRAP)/MyD88-adaptor-like (MAL), TIR-domain-containing
adaptor protein-inducing IFN-b (TRIF)/TIR-domain-containing molecule 1 (TICAM1)
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and TRIF-related adaptor molecule (TRAM; Oshiumi et al., 2003; Yamamoto et al., 2002;
Figure 1.4).
Figure 1.4. TLR signaling in conventional dendritic cells, macrophages and plasmatic dendritic cells.
Abbreviations: IKK complex, Inhibitor of nuclear factor kappa-B kinase complex; IKKα, Inhibitor of nuclear factor kappa-B kinase subunit alpha; IRAK4,1,2, Interleukin-1 receptor-associated kinase 4, 1, 2;
IRF3, 7, Interferon regulatory factor 3, 7; MAP kinase, Mitogen-activated protein kinase; MyD88, myeloid differentiation factor 88; NFkB, Nuclear-factor kappa B; RIP1, receptor-interacting protein 1; TLR, Toll- like receptor; TAK1, Transforming growth factor beta-activated kinase 1; TBK1/KKi, kinase binding domain; TIRAP, Toll/interleukin-1 receptor domain-containing adapter protein; TRAM, Translocating chain-associated membrane protein 1; TRAF3,6, TNF receptor-associated factor 3; TRIF, TIR-domain- containing adapter-inducing interferon-β (modified from Kumar et al., 2009).
